Optically active substrates

ABSTRACT

A device ( 10 ) for optical examination of biological materials ( 68 ) using radiation of a selected wavelength includes a substrate ( 50 ) having a first surface and a second surface opposite to the first surface. The first surface includes a dense array of micro-optical elements ( 54 ) arranged to provide increased intensity radiation or evanescent radiation. The first surface is in close proximity to the biological material ( 68 ) being examined.

BACKGROUND OF THE INVENTION

[0001] This application claims priority from U.S. ProvisionalApplication Ser. No. 60/213,987, filed on Jun. 25, 2000, entitledOptically Active Substrates, which is incorporated by reference.

[0002] The present invention relates to detecting and analyzingbiological materials by optical scanning, and particularly relates tooptical scanning or imaging of biological materials located on opticallyactive substrates.

[0003] Microarray technology enables studying complex biochemicalreactions and systems at once instead of studying them individually. Thetechnology provides a massively parallel form of analysis that increasesdata collection per unit time, decreases the overall time required foranalysis, uses smaller sample volumes and reagent volumes and sometimesreduces disposable consumption. Although the initial cost may be high,overall the technology represents considerable savings in the time andcosts of associated labor. Microarray technology became a fundamentaltool for genomic research. The technology can also be utilized forroutine analysis used in clinical diagnostics or for industrialanalytical purposes.

[0004] In general, microarrays can be created by optical (or otherradiation) directed synthesis, or by microfluidic delivery of nucleicacids onto different substrates. The first technique usesphotolithography or other submicron technologies to define positions atwhich single specific nucleotides are added to growing single-strandednucleic acid chains. Series of precisely defined nucleotide additionsand light directed chemical linking steps are used to synthesizehigh-density arrays of defined oligonucleotides on a solid substrate. Amicroarray of probe sequences may be fabricated by using techniquesdescribed in U.S. Pat. No. 5,143,854 or PCT Application published as WO92/10092, or U.S. Pat. Nos. 5,384,261; 5,405,783; 5,412,087; 5,424,186;5,445,934; 5,744,308 all of which are incorporated by reference.Alternatively, microarrays can be fabricated by other techniques asdescribed in PCT Application PCT/US99/18438 published as WO 00/09757,which is incorporated by reference.

[0005] According to a second technique, microarrays are created bymicrofluidic delivery, as described in PCT Application PCT/US99/00730,published as WO 99/36760, which is incorporated by reference. Thesemicroarrays can contain a wide range of biological materials including,plant, animal, human, fungal and bacteria cells; viruses, peptides,antibodies, receptors, and other proteins; cDNA clones, DNA probes,oligonucleotides, polymerase chain reactions (PCR) products, andchemicals. These biological materials are delivered in form of an arrayof spots to various microarray substrates including chemically treatedglass microscope slides, coverslips, plastics, membranes, or gels. Thenumber of deposited spots is in the range of 100 to 50,000 permicroarray, and the diameter of an individual spot is in the range of 50μm to 1000 μm, and preferably 100 μm to 250 μm. The volume of eachdeposited spot is in the range of 10 pL to 10 nL, and preferably 50 pLto 500 pL.

[0006] There are several types of microarray scanning and imagingsystems for viewing the entire microarray. Myles et al. describeddifferent optical scanning and imaging systems in Microarray BiochipTechnology, pp. 53-64, edited by M. Schena, published by BioTechniquesBooks, Natick, Mass. These scanning systems utilize XYZ stage scanningwith a stationary microscope objective, pre-objective scanning, orflying objective scanning with a translation stage. Some fluorescencemicroscopes use a CCD array imager as a detector. Numerous examples aredescribed in the Handbook of Biological Confocal Microscopy edited byJames Pawley, Plenum Press, 1989 and 1995, or by Sampas in U.S. Pat. No.5,900,949.

[0007] Many of the above-mentioned instruments are epifluorescentpseudo-confocal laser scanning microscopes. They are pseudo-confocalsystems because they have two optical paths. The first path is theexcitation path (also called laser path) that defines the pixel size,typically between 3 and 10 μm. This path has a relatively low numericalaperture, around 0.1, and consequently a greater depth of field. Thesecond path is the emission path (also called detection path), designedto maximize energy collection and therefore has the highest possiblenumerical aperture; however, this provides a small depth of field. Atrue confocal microscope uses the same numerical aperture for bothlenses and a relatively small depth of field, which is used to create“optical sections” of three-dimensional structures. The confocalmicroscopy is used to reject out of focus background signal.

[0008] The depth of field expresses the axial tolerance in locating thesample in order to obtain a valid measurement of a picture element(i.e., a pixel) needed to construct an image. The depth of field is alsodetermined by the necessity to accommodate some irregularities of thelarge size of the slide or sample inspected. The depth of field (DOF) ofa microscope is a function of the energy collection capability of theobjective lens as defined by its numerical aperture (NA) and the lightwavelength (λ), wherein DOF≈λ/(NA)². The desired resolution of an imagedepends on the spot size (d) of the optical system expressed as afunction of the numerical aperture (NA) of a perfect objective and thelight wavelength (λ), wherein d≈λ/NA.

[0009] The energy an optical system gathers in order to have ameaningful signal from the detector is expressed at a firstapproximation by the second power of the numerical aperture of theobjective lens. The numerical aperture is a function of the lensgeometry and the index of refraction (n) of the medium, i.e., NA=n sinθ, where θ is the angle formed by the radius of the lens and its focaldistance, and n=1 in air. For example, for an objective with NA=0.7 usedwith visible light, DOF is about 2 micron; however, an objective withNA=0.2 collects less than about {fraction (1/10)} of light as theobjective having NA=0.7, but it has DOF=37 microns.

[0010] Practical considerations for microarray scanning require a largedepth of field to accommodate a lens, a scanning stage, substrateflatness and tolerances, and other imperfections. Consequently, therehas to be a compromise between the amount of light collected (i.e., thesize of NA) and the required depth of field. That is, microarrayscanning and imaging instruments require several performance trade-offs.

[0011] Fluorescence microscopy is a relatively inefficient process,wherein the light source-to-detector efficiency is estimated in partsper trillions. There is usually a very low efficiency of thefluorescence conversion. Furthermore, among other limitations, thescanning systems cannot increase the intensity of the illumination bythe laser source, because the fluorescent sample would be destroyed;this is known as photo-bleaching. Also, before photo-bleaching takesplace, most fluorophores behave in a non-linear and possiblyunpredictable manner. Additionally, numerous non-optical constrains comeinto play such as acceptable scan duration, detector performance, andelectronic and image manipulation processes.

[0012] Therefore, there is a need for optical scanning or imagingsystems with high source-to-detector efficiency and for samplesubstrates and packaging that efficiently utilize light for opticalscanning and imaging.

SUMMARY OF THE INVENTION

[0013] The present invention relates to a system, product, and methodfor detecting and analyzing biological materials by optical scanning.The present invention utilizes optically active substrates that are usedwith various probes for detecting or analyzing biological material suchas polymers. The optically active substrates may also provide anoptically cooperating support for arrays of polymer sequences, such asoligonucleotide arrays.

[0014] According to one aspect, a device for optical examination ofbiological material using radiation of a selected wavelength, includes asubstrate having a first surface and a second surface opposite to thefirst surface. The first surface comprises a dense array ofmicro-optical elements being arranged to form “increased intensity”radiation near the elements. The first surface is in close proximity tobiological material being examined.

[0015] According to another aspect, a wide field of view, scanningmicroscope for examination of biological material on a first surface ofan optically active substrate comprises a scanning assembly for anobjective lens. The scanning assembly includes a support structureassociated with a driver and constructed to travel in a periodic motionover the substrate in a predetermined linear or arcuate scan path. Theobjective lens delivers light for essentially on-axis scanningthroughout a scan range of the assembly. The driver for the supportstructure is adapted to displace the support structure. The objectivelens also collects light from the optically active substrate.

[0016] Preferred embodiments of these aspects include one or more of thefollowing features. The second surface of the device (i.e., theoptically active substrate) is oriented for receiving the radiationemitted from a light source of a scanning microscope. The second surfaceof the device is also oriented for providing radiation to a detector ofa scanning microscope after interaction of the increased intensityradiation with the biological material.

[0017] The micro-optical elements are, for example, semi-spherical,aspherical, semi-conical, semi-hyperbolic, semi-parabolic orsemi-triangular micro-lenses. Alternatively, the micro-lenses are formedby micro-cavities having parallel or semi-parallel groves in the form ofhalf cylinders, quarter cylinders, cones, spheres, triangles,hyperbolas, ovaloids, or other geometrical shapes. The micro-lenses arepreferably formed by micro-cavities formed inside the substrate.

[0018] The micro-cavities may be formed inside the substrate byspherical indentations of approximately one radius or a fraction ofradius in depth. There are numerous methods for forming themicro-cavities or micro-structures including molding, hot pressing orother. The micro-optical elements may include a grating or teeth-likestructures.

[0019] The optically transparent substrate has a thickness between thefirst and second surface of about 1 mm. Each said micro-optical elementhas a dimension comparable to, or somewhat larger or smaller than thewavelength of the radiation. The surface of the micro-optical elementsmay include a layer of a high index medium transparent at the employedwavelengths. The high index medium is deposited by one of the following:sputtering, evaporation, or MOCVD.

[0020] The micro-optical elements may include high density micro-lenseshaving a high index of refraction. The high density micro-lenses withthe high index of refraction are made by vacuum deposition onto thefirst surface.

[0021] The micro-optical elements may be micro-lenses or other elementsformed inside or on the surface of the substrate, having a radius orother periodic dimension in the range of 0.1 μm to 10 μm, or for somestructures less than 100 μm. Preferably, the dimensions are comparableto the wavelength.

[0022] The optically transparent substrate can be made of one of thefollowing: polycarbonate disc, Mylar® based disc, PMMA disc, Plexiglas®disc or similar plastic disc with an index of refraction about 1.57. Theoptically transparent substrate can be also made of glass or quartz.

[0023] The first surface is arranged to support a probe array. The firstsurface is arranged to support fluorescently labeled biologicalmaterial. Then, the substrate is made of a material transparent tofluorescent light emitted from fluorophores excited at their specificemission wavelength.

[0024] The high index coating is made of titanium dioxide with an indexof refraction of about 2.4, gallium phosphate with an index ofrefraction of about 3.4 or other medium with suitable index andtransmission coefficient at the wavelength. The high index coating isdeposited by one of the following: sputtering, evaporation or MOCVD. Thehigh index coating has a thickness in the range of about 10 nm to 1000μm, and preferably in the range of about 0.1 μm to 10 μm, depending onthe material. Preferably, the material has a thickness that causes lowattenuation, i.e., acceptable optical losses, since these coatings havea relatively low coefficient of transmission.

[0025] The optically active substrates are used for scanning ofdeposited or attached biological material. The optically activesubstrates can support thin tissue sections (processed by washing awaysome of the tissue and other methods known in the art.) The opticallyactive substrates can also support oligonucleotide spots or featuresarrayed on a uniform featureless flat hard or soft, porous, ornon-porous material.

[0026] According to another preferred method, fluorescently labeledbiological material is deposited on the optically active substrate. Anoptical system images the deposited biological material. An emittedexcitation beam is delivered by an optical element (e.g., an objective)to illuminate a number of microlenses (or other optically activeelements) that “focus” or “intensify” the excitation beam. This type of“intensified” radiation or evanescent radiation excites fluorophoresthat emit light at their specific emission wavelength. The emittedfluorescent radiation is detected by a detector.

[0027] The optical system may include several embodiments. For example,the emitted fluorescent radiation may be collected by the microlenses(or other types optically active elements located on the opticallyactive substrate) and transmitted back through the substrate. In thisarrangement, a detector receives the transmitted fluorescent radiationvia an objective lens (or optical system) located in a reflectiongeometry. Alternatively, the emitted fluorescent radiation is collectedby a lens (or an optical system) located in a transmission geometry. Inthis embodiment, the detector receives fluorescent (or non-fluorescent)radiation that doesn't travel back through the substrate. In thetransmission geometry, the microlenses (or other types optically activeelements) do not “intensify” the fluorescent radiation, but thisradiation is not attenuated by the optically active substrate.

[0028] According to yet another aspect, a high-resolution epifluorescent confocal microscope is made with extremely high numericalaperture (possibly as high as NA=3) with a sub-micron resolution and anextremely great depth of field.

[0029] According to yet another aspect, the optically active substrateis used with a chip cartridge (or chip package) described in U.S. Pat.No. 5,945,334.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 is a schematic illustration of an optical scanning andimaging system.

[0031]FIG. 1A is a schematic illustration of another optical scanningand imaging system.

[0032]FIG. 2 shows diagrammatically a beam scanning system and a flowsystem used in the optical scanning and imaging system of FIG. 1A.

[0033]FIG. 2A is a detailed cross-sectional view of a flow cell usedwith an optically active substrate, as shown in FIG. 2.

[0034]FIG. 3 shows an enlarged cross-sectional view of an opticallyactive substrate.

[0035]FIG. 4 shows an embodiment of a chip cartridge used forexamination of biological material.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036]FIG. 1 illustrates one embodiment of a confocal optical scanningand imaging system for examination of biological material located on, ornear, an optically active substrate shown in FIG. 3. The opticallyactive substrate forms an important part of the optical system designedfor increasing the signal-to-noise ratio of the detected optical signal.

[0037] Referring to FIG. 1, optical system 10 includes a light source12, an entrance aperture 14, a lens 16, a dichroic mirror 20, anobjective lens 24, a two or three axis translation table 28, a lens 32,an exit pinhole 34, a band pass filter or a rejection filter 31 and adetector 36. In the following embodiment, optical system 10 is arrangedfor the detection of fluorescent light; however, optical system 10 mayalso be arranged for the detection of scattered or transmitted light atthe irradiation wavelength.

[0038] Light source 12 emits an excitation light beam 15, and dichroicmirror 20 directs the excitation light toward objective lens 24.Objective lens 24 focuses light onto a pixel (A) located on or near anoptically active substrate 26. Fluorescent light emitted from pixel A iscollected by objective lens 24 and transmitted through dichroic mirror20, over a light path 30, toward and trough band pass or rejectionfilter 31 and to light detector 36.

[0039] The arrangement of apertures (pinholes) 14 and 34 and lenses 16and 32 provides to detector 36 fluorescent light from a selected depth(in the Z direction) of pixel A. At pixel A, light emitted from otherdepths in the Z direction is substantially blocked and doesn't passthrough pinhole 34. This spatial filter improves the signal-to-noiseratio, which is known in the art.

[0040] Light source 12 is constructed to emit light of a wavelengthcapable of exciting fluorophores associated with the examined biologicaltissue located on the optically active substrate. As shown in FIG. 3,optically active substrate 50 is transparent and includes opticallyactive surface 52 with a dense array of micro-optical elements 54.Micro-optical elements 54 are arranged to support biological material68. Micro-optical elements 54 may be formed by microlens cavities 56 orother optical elements located on surface 52.

[0041] Referring to FIGS. 1 and 1A, for example, light source 12 emitssimultaneously or sequentially 473, 488 or 490 nm (or 532 nm, 638 nm or745 nm) light directed to optically active substrate 26 by dichroicmirror 20. For example, the excitation light of 488 nm irradiates apixel on the surface of substrate 26, and excites fluorophores that emitfluorescent light, for example, in the range 515 nm to 595 nm. Theexamined biological tissue can be labeled using various types offluorophores (and their corresponding absorption maxima) are Fluorescein(488 nm), Dichloro-fluorescein (525 nm), Hexachloro-fluorescein (529nm), Tetramethylrhodamine (550 nm), Rhodamine X (575 nm), Cy3™ (550 nm),Cy5™ (650 nm), Cy7™ (750 nm), and IRD40 (785 nm). Detector 36 uses asuitable band pass or rejection filters for detecting the fluorescentlight emitted from pixel A. Preferably, objective lens 24 has a largenumerical aperture (a numerical aperture of at least above 0.1). Opticalsystem 10 can collect optical data over an array of pixels by displacingoptically active substrate 26 (or optically active substrate 50) in theX and Y directions using a translation table 28.

[0042] In general, light source 12 may be a lamp with a filter or alaser (solid state or gas laser such as an argon laser, a helium-neonlaser, a diode laser, a dye laser, a titanium sapphire laser, afrequency-doubled diode pumped Nd:YAG laser, or a krypton laser).Typically, the excitation source illuminates the sample with anexcitation wavelength that is within the visible spectrum, but otherwavelengths (i.e., ultraviolet or near infrared wavelengths) may be useddepending on the type of markers or samples or detection methods. Lightdetector 36 may be a photomultiplier (PMT), a diode, a CCD array, oranother photodetector.

[0043]FIG. 1A is a block diagram of an optical scanning system 40, whichincludes at least one light source, several optical detectors, a lightpath system for providing optical coupling, a fast scanning system, anda system controller. The light path system may include dichroicbeamsplitters, spectral filters, pinholes and several channels fordetecting wavelength specific radiation. The optical scanning system 40also uses the novel optically active substrates described in detail inconnection with FIG. 3.

[0044] The optically active surface has a dense array of micro-opticalelements, each having a dimension comparable to (or larger than) thewavelength of the radiation emitted from the light source. Themicro-optical elements are constructed and arranged to generateincreased intensity radiation or excite an evanescent radiation in theirclose proximity. The optically active surface may be combined with othertechniques for examination of biological material, which techniques arementioned and incorporated by reference above. Typically, opticalscanning system 10 or 40 is used to obtain images of oligonucleotidemicroarrays to which fluorescently labeled DNA or RNA is bound, imagesof polypeptides or other polymer arrays, electrophoresis gels, or otherbiological specimens. The optically active surface improves theefficiency of the detection process.

[0045] Referring to FIG. 1A, a controller 85 controls the entireoperation of optical scanning system 40 including a beam scanner 48.Scanning system 40 specifically includes a laser 12 for providingradiation of a selected wavelength to a stationary light path system 12.In the stationary light path system, the emitted beam 41 is partiallyreflected and partially transmitted by a beamsplitter 42. The reflectedportion of beam 41 impinges upon a photodetector 43B (optional), whichis typically a photodiode used for laser power monitoring. Thetransmitted portion of beam 41, traveling over a light path 44, isreflected by a dichroic beamsplitter 45, transmitted through a lens 46and an aperture (pinhole) 47, expanded to the desired diameter, anddelivered to a beam scanner 48.

[0046] The beam scanning system, schematically shown as box 48, includesseveral optical elements arranged for scanning a sample that may belocated on a linear translation stage. Beam scanning system 48 deliverssequentially a focused light beam to a series of pixels, and conveysreflected or fluorescent light from each pixel back to the light pathsystem. Beam scanning system 48 has several high scan rate embodimentsdiscussed in detail below.

[0047] Beam scanning system 48 also provides the “return” light path(shown in FIGS. 2 and 3) for light re-emitted from a sample, and focusesthe re-emitted light onto confocal pinhole 47. Light transmitted throughaperture 47 is collimated by lens 46. For example, light re-emitted fromthe sample having wavelengths less that 515 nm is reflected bybeamsplitter 45 and partially reflected by beamsplitter 42 to arrive atphotodetector 43A. (Alternatively, pinhole 47 may be located in a lightpath 101 to spatially filter only the fluorescent radiation). Lighttransmitted through beamsplitter 45 travels over light path 101 to thefour optical channels 90, 100, 110 and 120 depending on its wavelength.The number of optical channels used in scanning system 40 is optional.For example, light of a wavelength between 515 μm and 545 nm, reflectedby a dichroic beamsplitter 102, passes through a filter 104 onto aphotodetector 106. Light reflected by a dichroic beamsplitter 112,having a wavelength between 545 nm and 570 nm passes through a filter114 and onto a photodetector 116. Similarly, a photodetector 126 detectslight having wavelengths between 570 nm and 595 nm, which passes througha filter 124. Light of wavelengths greater than 595 nm passes through afilter 94 onto a photodetector 96. Preferably, photodetectors 96, 106,116, and 126 are photomultipliers (PMTs). Each optical channel 90, 100,110 and 120 may include a confocal pinhole adjacent to a lens (not shownin FIG. 1). Confocal pinhole transmits florescence originating from thefocal plane of system 10.

[0048] Referring to FIG. 2, beam scanning system 48 may include a flyingobjective design (an oscillatory movement design or a linear movementdesign), a scanning XY design, or preobjective scanning design. Thesedesigns are diagrammatically shown in FIG. 2 as a box 60 (e.g.comprising a flying objective, a scanning mirror etc.). Beam scanningsystem 48 provides an irradiation beam 49 directed toward an opticallytransparent substrate 50 mounted on a flow cell 70. For example, flowcell 70 is located on an XYZ stage 80 controlled by a controller 85(which can also control the entire scanning system 40 and severalelements of a hybridization system). Flow cell 70 includes a body havinga cavity 71, which is about 50 μm to 1500 μm deep, having the bottom andsides of cavity 71 preferably light absorbing. Flow cell 70 includes aninlet port 73 and an outlet port 74.

[0049]FIG. 2A is a cross-sectional view of flow cell 70 with substrate50 mounted on flow cell 70 using adhesive bonds 72 or by any otherdesign. Substrate 50 has an optically inactive surface 51 and anoptically active surface 52. Reagents, such as labeled targets areinjected into the cavity 71 through inlet port 73 by a pump 75 (FIG. 2)or by using a syringe, as described in U.S. Pat. No. 5,631,734, which isincorporated by reference. Within cavity 71, the reagents bind with oneor more complementary probes located on surface 52 (located insidecavity 71 in the embodiment of FIG. 2). The reagents are circulated intothe cavity via inlet port 73 by pump 75 and exit through outlet port 74for re-circulation or disposal. A user can change hybridizationconditions without removing substrate 50. Flow cell 70 may also includea temperature controller connected to a re-circulating bath device 76,as described in U.S. Pat. No. 5,631,734. Flow cell 70 may also includean agitation system and several valves, containers and cavities asdescribed in U.S. Pat. Nos. 5,945,335 and 6,140,044, which areincorporated by reference.

[0050] Referring to FIG. 3, optically transparent substrate 50 hasoptically active surface 52 with a dense array of micro-optical elements54. Micro-optical elements 54 may be formed by microlens cavities 56 orany other optical elements that provide “increased intensity” radiationor “evanescent” radiation. In the embodiment of FIG. 3, micro-opticalelements 54 include microlens cavities 56 with a high index film(coating or layer) 58 deposited by sputtering or another vacuumdeposition.

[0051] Microlens cavities 56 have a radius or a width (section oranother relevant dimension) in the range of 100 nm to 1 μm; that is, afraction or a small multiple of the wavelength of interest. The diameterand depth of the microlens cavities may define the thickness of the highindex layer 58, which may even completely fill the cavities. High indexlayer 58 is made of titanium dioxide with an index of refraction of 2.4,gallium phosphate with an index of refraction 3.4, or other medium withsuitable index of refraction. High index layer 58 is designed to have athickness depending on its transmission coefficient so that a relativelylow transmission coefficient will cause acceptable optical losses.Alternatively, micro-optical elements 54 are formed by parallel orsemi-parallel groves, parallel or semi-parallel half cylinders, or othermicro-structures.

[0052] Substrate 50 is made of a PMMA, Plexiglas® or a similar plasticwith an index of refraction about 1.57. The microlens cavities could becreated in the substrate by forced embossing at a proper temperature, orby casting against a suitably formed negative master (as used whencreating CD and DVD discs). Alternatively, substrate 50 is be made byetching, which is preferably done for glass or quartz.

[0053] Micro-optical elements 54 may perform one or several functions.Micro-optical elements 54 may launch an evanescent surface wave 78 (FIG.3) within biological material 68 (e.g., a spot of dehydrated or hydratedbiological material). Micro-optical elements 54 may also serve as highNA optical element capable of gathering light emitted when theexcitation radiation of the surface wave excites a fluorophore 69located within or at the biological material. The micro-optical elementsexhibit an efficient light gathering effect since they behave as alenslets (or other optical elements) with a very high numericalaperture.

[0054] The shape of micro-optical elements 54 and the selection of therefraction index of substrate 50 permit the fluorescent emission from afluorophore 69 to egress from the exposed surface of the support with acomparatively low divergence and preferably in a collimated manner, asshown by lines 67 in FIG. 3.

[0055] In the embodiment of FIG. 3, the emitted fluorescent radiation iscollected by micro-optical elements 54 and transmitted back through thesubstrate. In this arrangement, a detector receives the transmittedfluorescent radiation via an objective lens (or optical system) locatedin a reflection geometry. Alternatively, the emitted fluorescentradiation is collected by a lens (or an optical system) located in atransmission geometry. In this embodiment, the detector receivesfluorescent (or non-fluorescent) radiation that doesn't travel backthrough the substrate. In the transmission geometry, micro-opticalelements 54 do not “intensify” the fluorescent radiation, but thisradiation is not attenuated by the optically active substrate.

[0056] Referring to FIG. 1A, according to a first embodiment, scanningsystem 48 includes an oscillatory or rotary design for displacing anobjective lens, for example, as described in PCT ApplicationPCT/US99/06097 (published as WO 99/47964) or U.S. ProvisionalApplication 60/286,578, both which are incorporated by reference. Therotary design caries a relatively simple and light objective lens, whichis oscillated over the substrate area. The objective lens delivers anexcitation light beam to an array of pixels of the scanned sample. Therotary design includes a periscopic structure that optically couples thescanning objective lens to the irradiation and detection light pathsshown in FIG. 1.

[0057] In this embodiment, the rotary architecture offers both highspeed scanning and a constant optical path length. The arc scanned bythe objective lens covers the width of substrate 50 in polarcoordinates. Substrate 50 is mounted on a translation stage for lineardisplacement. The data, acquired in polar coordinates for each slideposition, is instantly converted to Cartesian coordinates by theinstrument's computer so the image is then directly correlated with themicroarray on substrate 50. The objective lens, carried on the rotarysupport structure may be an aspheric, one-element lens. Advantageously,the field of view is always on axis, which eliminates all the commonsources of lateral or chromatic aberrations found in preobjectivescanner microscopes.

[0058] According to another embodiment, beam scanning system 48 mayinclude a rectilinear flying objective design, as described, in U.S. PatNo. 5,459,325 to Hueton et al., which are incorporated by reference. Therectilinear flying objective design includes a lens mounted on a lineararm driven by a voice coil to perform a fast scan over sample substrate50 in a first dimension. Substrate 50 is mounted on a translation stagethat displaces sample in a second dimension, and may also move substrate50 in a third dimension (i.e., z direction) for focusing.

[0059] The rectilinear flying objective design uses a reliable,low-cost, low-inertia, stable high-speed linear scanning system, whichdirectly acquires the data in the Cartesian coordinate system. Theoptical data may be acquired in both directions of the lens scan, thusdoubling the effective scanning speed. The scanning system includes anstable and rigid structure that enables high frequency scanning.

[0060] According to another embodiment, beam scanning system 48 mayinclude a preobjective scanning design described in U.S. Pat. No.5,981,956 to Stern or in U.S. Pat. No. 5,631,734 to Stern et al., bothof which are incorporated by reference. The preobjective scanning designuses a scanning mirror that scans the excitation beam over a large fieldof view objective that includes several elements to provide, forexample, a 10 mm field of view. This large field of view objective has acomparatively low NA of about 0.25. The examined substrate is translatedlinearly under the objective whiled the excitation beam is scanned overthe objective by a scanning mirror. To scan an area larger than 10 mmwide, the optically active substrate can be translated sideways andscanned to capture a second swath. The two swaths are later stitchedtogether by the instrument's computer. The major benefit of lownumerical aperture (e.g., NA=0.25) is a large depth of field (e.g., 16μm), which accommodates imperfections.

[0061] According to another embodiment, optical scanning system 10 maybe replaced by an imaging system that uses a CCD array as described, forexample, in U.S. Pat. No. 5,578,832, which is incorporated by reference.

[0062] Scanning and imaging systems 10 and 40 achieve a uniformperformance (i.e., consistent data) over the entire surface of substrate50. The intensity uniformity is about 95% and the spatial uniformity isabout 98%. The system scan over a selected scan area (typically over ascan area of 20 mm×65 mm) and have a resolution of about 2.0 μm to 4.0μm, wherein 10 μm is a sufficient resolution for a 10 μm spot size.

[0063] As known in the art and described, for example, in U.S. Pat. No.5,910,940 an evanescent field arises at the boundary between a highrefractive index medium and an adjacent lower index medium due to totalinternal reflection, as where the parent field in the higher indexmedium penetrates into the lower index medium (i.e., the refractionangle becomes imaginary). This evanescent field is a continuation of theinternal standing wave that in turn is a result of constructiveinterference of incident and reflected illumination at the interface.(In the quantum mechanical view, this penetration of the totalreflection barrier is called photon tunneling.) Immediately at theactive layer (or low-index side of medium interface), the resultantintensity can be several times larger than the intensity of the incidentradiation.

[0064] Evanescent field also arises when propagating illumination isdiffracted by a grating having the grating period smaller than thewavelength, such that the diffracted orders are evanescent (i.e., thediffraction angle becomes imaginary). Again, the resultant intensity canbe several times larger than the intensity of the incident radiation.

[0065] The amplitude of evanescent field decays exponentially withdistance from the surface of the medium interface. The exponent of thedecay depends on the ratio of denser to lower indices of refraction atthe boundary surface. Thus, evanescent field decays over a distance ofonly less than a micron; thus this is “near-field.” Near field includesboth propagating and non-propagating radiation near, that is, within awavelength of the interface surface. For interaction with biologicalmedium 68, biological medium 68 has to be located close to the surfacewith the high-refractive index boundary (interface of the dielectricmaterial), or close to the diffraction grating.

[0066] To increase the strength of the created evanescent radiation,higher refractive index medium is required. The index of refraction ofavailable materials imposes a practical limit of about 2.4 in thevisible spectrum, and about 3.5 in the near infra-red spectrum.Conversion by diffraction is limited only by the diffraction gratingspatial period. The present embodiments can use a diffraction gratinghaving the spatial period of less than 40 nanometers.

[0067] Different types of substrates have been used to excite evanescentradiation for use in microscopy, as described in U.S. Pat. No.5,633,724; 5,437,840; or U.S. Pat. No. 5,351,127 to King or U.S. Pat.No. 5,341,215 to Seher, or European Patent Application 93304605.4 (EP0575 132 A1) by King.

[0068] Alternatively, optically active substrate 52 includes aninterference grating buried under a small layer of high index glass.This optically active substrate 52 is a modification of microscopeslides described in U.S. Pat. Nos. 5,822,472 and 5,959,292 (which areincorporated by reference), and are available from Zeptosens AG(Witterswill Switzerland). The grating diffracts the incident lightand/or light that has not been absorbed by the examined biologicalmaterial (which is a very large fraction) and induces, at a suitableangle, an evanescent wave, which then interacts with the biologicalmaterial. The intensity of the excited evanescent wave can be one orderof magnitude greater than that of the original incident light beam.Since the grating reflects the beam at a different angle for eachwavelength, each slide can operate beneficially at their designedwavelength.

[0069]FIG. 4 show an embodiment of a cartridge 150 for packaging andhandling substrate 50. Cartridge 150 includes a top casing 152, a middlecasing 154 and a bottom casing 156 made of a plastic material andarranged to encase a hybridization cavity. Bottom casing 156 includestwo fluid channels arranged in communication with two annular regionslocated in middle casing 154. Middle casing 14 includes a septum (or thelike) located at the annular regions and arranged to seals fluids withintwo channels located in top casing 154 and in communication with thehybridization cavity, as described in detail in U.S. Pat. No. 5,945,334,which is incorporated by reference. Chip package 150 also includesalignment holes 158 and 160 and several support structures for mountingor positioning the chip package relative to a scanner.

[0070] The probe arrays may be fabricated according to varioustechniques disclosed in U.S. Pat. No. 5,143,854 to Pirrung et al., PCTApplication WO 92/10092, or U.S. Pat. Nos. 5,384,261; 5,405,783;5,412,087; 5,424,186; 5,445,934; or 5,744,308 all of which areincorporated by reference. As described above, micro-optical elements 54provide high energy gathering in the proximity of the biologicalmaterial 68 located on the probe arrays.

[0071] In general, a probe is a surface-immobilized molecule that isrecognized by particular target and is sometimes referred to as aligand. Examples of probes that can be investigated by this inventioninclude, but are not restricted to, agonists and antagonists for cellmembrane receptors, toxins and venoms, viral epitopes, hormones (e.g.,opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes,enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotidesor nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.

[0072] A target is a molecule that has an affinity for a given probe andis sometimes referred to as a receptor. Targets may benaturally-occurring or manmade molecules. Also, they can be employed intheir unaltered state or as aggregates with other species. Targets maybe attached, covalently or noncovalently, to a binding member, eitherdirectly or via a specific binding substance. Examples of targets whichcan be employed by this invention include, but are not restricted to,antibodies, cell membrane receptors, monoclonal antibodies and antiserareactive with specific antigenic determinants (such as on viruses, cellsor other materials), drugs, oligonucleotides or nucleic acids, peptides,cofactors, lectins, sugars, polysaccharides, cells, cellular membranes,and organelles. Targets are sometimes referred to in the art asanti-probes or anti-ligands. As the term “targets” is used herein, nodifference in meaning is intended. A “probe target pair” is formed whentwo macromolecules have combined through molecular recognition to form acomplex.

[0073] Substrate 50 is preferably optically transparent, but it does notneed to be optically transparent, for example, if used having opticallyactive surface 52 facing incoming radiation beam 46. Substrate 50 may befabricated of a wide range of material, either biological,nonbiological, organic, inorganic, or a combination of any of these,existing as particles, strands, precipitates, gels, sheets, tubing,spheres, containers, capillaries, pads, slices, films, plates, slides,etc. The substrate may have any convenient shape, such as a disc,square, sphere, circle, etc. The substrate is preferably flat but maytake on a variety of alternative surface configurations. For example,the substrate may contain raised or depressed regions on which a sampleis located. The substrate and its surface preferably form a rigidsupport on which the sample can be formed. The substrate and its surfaceare also chosen to provide appropriate light-absorbing characteristics.For instance, the substrate may be a polymerized Langmuir Blodgett film,functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon,or any one of a wide variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,polycarbonate, or combinations thereof. Other materials with which thesubstrate can be composed of will be readily apparent to those skilledin the art upon review of this disclosure.

[0074] While the invention has been described with reference to theabove embodiments, the present invention is by no means limited to theparticular constructions described and/or shown in the drawings. Thepresent invention also comprises any modifications or equivalents withinthe scope of the following claims.

1. A device for optical examination of biological material usingradiation of a selected wavelength, comprising an optically transparentsubstrate having a first surface and a second surface opposite to thefirst surface, the first surface comprising a dense array ofmicro-optical elements being arranged to generate increased intensityradiation near the optical elements, the first surface being in closeproximity to the examiner biological material.
 2. The device of claim 1wherein the second surface is oriented for receiving the radiationemitted from a light source of an optical system.
 3. The device of claim2 wherein the second surface is oriented for providing radiation to adetector of an optical system after interaction of the increasedintensity radiation with the biological material.
 4. The device of claim1 wherein each said micro-optical element has a dimension comparable tothe wavelength of the radiation.
 5. The device of claim 1 wherein themicro-optical elements are micro-lenses.
 6. The device of claim 5wherein the micro-lenses are formed by micro-cavities formed inside thesubstrate.
 7. The device of claim 5 wherein the micro-lenses are formedby micro-cavities having parallel or semi parallel groves in the form ofhalf cylinders or quarter cylinders.
 8. The device of claim 6 whereinthe micro-cavities are formed inside the substrate by sphericalindentations one radius in depth.
 9. The device of claim 1 wherein themicro-optical elements comprise a grating.
 10. The device of claim 1wherein the micro-optical elements are teeth-like structures.
 11. Thedevice of claim 1 wherein the optically transparent substrate has athickness between the first and second surface of about 1 mm.
 12. Thedevice of claim 1 wherein a surface of the micro-optical elementsincludes a layer of a high index medium transparent at the wavelength.13. The device of claim 12 wherein the high index medium is deposited byone of the following: sputtering, evaporation, MOCVD.
 14. The device ofclaim 1 wherein the micro-optical elements include high densitymicro-lenses having a high index of refraction.
 15. The device of claim14 wherein the high density micro-lenses has the high index ofrefraction are made by deposition onto the first surface.
 16. The deviceof claim 1 wherein the micro-optical elements are micro-lenses formed bymicro-cavities inside the substrate, the micro-cavities having a radiusin the range of 0.1 μm to 10 μm.
 17. The device of claim 1 wherein themicro-optical elements are micro-lenses formed by micro-cavities insidethe substrate, the micro-cavities having a radius less than 100 m. 18.The device of claim 1 wherein the micro-optical elements aremicro-lenses formed by micro-cavities inside the substrate and adiameter and depth of the micro-cavities define the thickness of a highindex coating deposited on the first surface.
 19. The device of claim 1wherein the optically transparent substrate is made of one of thefollowing: polycarbonate disc, Mylar based disc, PMMA disc, Plexiglasdisc or similar plastic disc with an index of refraction about 1.57. 20.The device of claim 1 wherein the first surface is arranged to support aprobe array.
 21. The device of claim 1 wherein the first surface isarranged to support fluorescently labeled biological material.
 22. Thedevice of claim 1 wherein the substrate is made of a materialtransparent to fluorescent light emitted from fluorophores excited attheir specific emission wavelength.
 23. The device of claim 18 whereinthe high index coating is deposited by one of the following: sputtering,evaporation or MOCVD.
 24. The device of claim 18 wherein the high indexcoating is made of titanium dioxide with an index of refraction of about2.4.
 25. The device of claim 18 wherein the high index coating is madeof gallium phosphate with an index of refraction of about 3.4 or othermedium with suitable index and transmission coefficient at thewavelength.
 26. The device of claim 18 wherein the high index coatinghas a thickness of about 10 angstrom to 1000 angstrom depending on thematerial so that a relatively low coefficient of transmission of thematerial causes acceptable optical losses.
 27. A wide field of view,scanning microscope for examination of biological material on a firstsurface of an optically active substrate comprises a scanning assemblythat includes a support structure associated with a driver andconstructed to travel in a periodic motion over the substrate in apredetermined path, said scanning structure including an objective lensfor delivering light for essentially on-axis scanning and for collectinglight from said optically active substrate.